microeco: An R package for ecological analysis of microbial community

Chi Liu

Aim

　R language and it’s packages ecosystem are wonderful tools for the data analysis. In community ecology field, many packages can be used for the ecological and evolutionary analysis, such as vegan[1], ape[2] and picante[3]. In microbial community ecology, with the development of the high-throughput sequencing techniques, the increasing data amount and complexity make the community data analysis a challenge. There has been a lot of R packages created for the community data analysis in microbial ecology, such as phyloseq[4], microbiomeSeq, ampvis2, mare and microbiome. However, we lack a flexible and modularized R package to analyze and manage all the data. Thus we created microeco R package for this goal. This package provide powerful plotting and statistical approaches.

Main Features

  • R6 Class to store and analyze data; fast, flexible and modularized
  • Plotting the taxonomic abundance
  • Venn diagram
  • Alpha diversity
  • Beta diversity
  • Differential abundance analysis
  • Effects of environmental variables on community structure
  • Network analysis
  • Null model analysis
  • Functional analysis

Class introduction

All the classes in microeco package depend on the R6 class. R6 uses the encapsulated object-oriented programming paradigm, which means that R6 is a profoundly different OO system from S3 and S4 because it is built on encapsulated objects, rather than generic functions. If you are interested in the class feature, read those (from book Advanced R):

  • A generic is a regular function so it lives in the global namespace. An R6 method belongs to an object so it lives in a local namespace. This influences how we think about naming. The methods belong to objects, not generics, and you call them like object$method().

  • R6’s reference semantics allow methods to simultaneously return a value and modify an object.

  • You invoke an R6 method using $, which is an infix operator. If you set up your methods correctly you can use chains of method calls as an alternative to the pipe. Every R6 object has an S3 class that reflects its hierarchy of R6 classes.

Details

microtable class

　Many tools can be used to do the bioinformatic analysis, such as QIIME[5], usearch(https://www.drive5.com/usearch/), mothur[6], and RDP(http://rdp.cme.msu.edu/). Although the format of results may be different from different tools, the main files can be classified as the following parts: 1-OTU table, the traditional species-sample table; 2-taxonomy table, the OTU taxonomy assignments information table; 3-phylogenetic tree. Generally, it is necessary to create a sample information table to store all the detailed sample information, including the physicochemical data and the missing value (NA). The funtions read_qiime_otu_table() and split_assignments() in package qiimer are appropriate for the parsing of raw QIIME OTU table file.

　We use microtable class to store all the required data for the following analysis. We use the example data in the package to show the tutorials. The dataset is the 16S sequencing results of wetland soils in China[7]. In this dataset, the env_data table is separate from the sample information table.

[1] “data.frame”

  101 102 103 104 105
OTU_4272 4 0 2 1 0
OTU_236 3 0 0 0 3
OTU_399 39 51 59 20 22
OTU_1556 10 8 10 4 8
OTU_32 37 41 3 19 39

[1] “data.frame”

Table continues below
  Kingdom Phylum Class
OTU_4272 k__Bacteria p__Firmicutes c__Bacilli
OTU_236 k__Bacteria p__Chloroflexi c__
OTU_399 k__Bacteria p__Proteobacteria c__Betaproteobacteria
OTU_1556 k__Bacteria p__Acidobacteria c__Acidobacteria
OTU_32 k__Archaea p__Miscellaneous Crenarchaeotic Group c__
  Order Family Genus Species
OTU_4272 o__Bacillales f__ g__ s__
OTU_236 o__ f__ g__ s__
OTU_399 o__Nitrosomonadales f__Nitrosomonadaceae g__ s__
OTU_1556 o__Subgroup 17 f__ g__ s__
OTU_32 o__ f__ g__ s__

[1] “data.frame”

SampleID Group Type Saline
1 IW NE Non-saline soil
2 IW NE Non-saline soil
3 IW NE Non-saline soil
4 IW NE Non-saline soil
5 IW NE Non-saline soil

[1] “data.frame”

Latitude Longitude Altitude Temperature Precipitation
52.96 122.6 432 -4.2 445
52.95 122.6 445 -4.3 449
52.95 122.6 430 -4.3 449
52.95 122.6 430 -4.3 449
52.95 122.6 429 -4.3 449

[1] “phylo”

Then, we make an object use microtable class. Indeed, this operation is very similar with that in package phyloseq[4], but this is simpler and faster.

[1] “microtable” “R6”

microtable class: sample_table have 90 rows and 4 columns otu_table have 14113 rows and 200 columns tax_table have 14113 rows and 7 columns phylo_tree have 14096 tips

To make the species and sample information consistent across different files within the dataset object, we can use function tidy_dataset() to trim the dataset.

microtable class: sample_table have 90 rows and 4 columns otu_table have 13628 rows and 90 columns tax_table have 13628 rows and 7 columns phylo_tree have 13628 tips

Then, we remove OTUs which are not assigned in the Kingdom "k__Archaea" or "k__Bacteria".

microtable class: sample_table have 90 rows and 4 columns otu_table have 13628 rows and 90 columns tax_table have 13330 rows and 7 columns phylo_tree have 13628 tips

We also remove OTUs with the taxonomic assignments “mitochondria” or “chloroplast”.

microtable class: sample_table have 90 rows and 4 columns otu_table have 13628 rows and 90 columns tax_table have 13296 rows and 7 columns phylo_tree have 13628 tips

Then, to make the OTUs same in otu_table, tax_table and phylo_tree, we use tidy_dataset() again.

microtable class: sample_table have 90 rows and 4 columns otu_table have 13296 rows and 90 columns tax_table have 13296 rows and 7 columns phylo_tree have 13296 tips

Then we use sample_sums() to check the sequence numbers in each sample.

[1] 10316 37087

Sometimes, in order to reduce the effects of species number on the diversity measurements, we need to perform the resampling to make the sequence number same across samples.

[1] 10000 10000

Then, we calculate the taxa abundance at each taxonomic rank using cal_abund(). This function return a list called taxa_abund containing several data frame of the abundance information at each taxonomic rank. The list is stored in the object microtable automatically.

[1] “list”

If you want to save the taxa abundance file to the computer, use save_abund().

Then, we calculate the alpha diversity. The result is also stored in the object microtable automatically. As an example, we do not calculate phylogenetic diversity.

We also calculate the beta diversity (distance matrix) using function cal_betadiv(). We provide four most frequently used indexes: Bray-curtis, Jaccard, weighted Unifrac and unweighted unifrac.

trans_abund class

　This class is used to transform abundance data for plotting the taxa abundance. We first use this class for the bar plot. We use the highest 10 Phylum in dataset.

We do not retain the sample names in x axis and add the facet to show group.

The groupmean parameter can be used to obtain the group-mean barplot.

The box plot is a good way to intuitionally show data distribution across groups.

We also show the heatmap using the high abundant Genus

trans_venn class

The trans_venn class is used for venn plotting. We first merge samples according to the “Group” column of sample_table.

When the groups are too many to show using venn plot, we can use petal plot.

Sometimes, we are interested in the particular and shared species. In another words, the composition of the particular or shared species may account for the different and similar parts of ecological characteristics across groups[8]. For this goal, we first transform the results of venn plot to the traditional species-sample table, that is, another object of microtable class.

[1] “microtable” “R6”

We use bar plot to show the composition at the Genus level.

We also try to use pie chart to show the composition at the Phylum level.

trans_alpha class

　Alpha diversity can be transformed and plotted using trans_alpha class. Creating this class can return two data frame-alpha_data and alpha_stat.

Group Measure N Mean SD SE
CW Observed 30 1843 220.6 40.27
CW Chao1 30 2553 338.1 61.73
CW ACE 30 2716 367 67.01
CW Shannon 30 6.308 0.5355 0.09777
CW Simpson 30 0.9897 0.01305 0.002382

Then, we test the differences among groups using the KW rank sum test and anova with multiple comparisons.

Groups Measure Test method p.value Significance
IW vs CW Observed KW 0.0371 *
IW vs TW Observed KW 0.4553
CW vs TW Observed KW 0.3912
IW vs CW vs TW Observed KW 0.155
IW vs CW Chao1 KW 0.002689 **
  Observed Chao1 ACE Shannon Simpson InvSimpson Fisher Coverage
IW a a a a a a a b
TW a ab b a a a a a
CW a b b a a a a a

Now, let us plot the mean and se of alpha diversity for each group, and add the agricolae::duncan.test result.

We can also use the boxplot to show the paired comparisons directly.

trans_beta class

　Beta diversity can be transformed and plotted using trans_beta class. The analysis referred to the beta diversity in this class mainly include ordination, group distance and manova. We first show the ordination using PCoA.

Then we plot and compare the group distances.

Clustering plot is also a frequently used method.

perMANOVA is often used in the differential test of distances among groups.

Permutation: free
  Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
Group 2 6.121 3.06 10.57 0.1955 0.001
Residuals 87 25.18 0.2895 NA 0.8045 NA
Total 89 31.3 NA NA 1 NA
Groups measure permutations R2 p.value Significance
IW vs CW bray 999 0.1595 0.001 ***
IW vs TW bray 999 0.147 0.001 ***
CW vs TW bray 999 0.1556 0.001 ***
Permutation: free
  Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
Group 2 6.121 3.06 12.01 0.1955 0.001
Type 3 3.783 1.261 4.949 0.1208 0.001
Residuals 84 21.4 0.2548 NA 0.6836 NA
Total 89 31.3 NA NA 1 NA

trans_diff class

　Differential abundance test is a very important part in the microbial community data analysis. It can be used to find the significant taxa in determining the community differences across groups. Currently, trans_diff class have three famous approaches to perform this analysis: metastat, rf and LEfSe[9]. Metastat[10] depends on the permutations and t-test and performs well on the sparse data. It is used for the comparisons between two groups.

LEfSe combines the non-parametic test and LDA and depends on the python. Now we rewrite this approach in this package.

Taxa Group pvalue LDA
k__Bacteria|p__Proteobacteria CW 2.004e-11 4.852
k__Bacteria|p__Acidobacteria IW 6.708e-12 4.786
k__Bacteria|p__Acidobacteria|c__Acidobacteria IW 6.47e-13 4.784
k__Bacteria|p__Bacteroidetes TW 5.139e-10 4.777
k__Bacteria|p__Proteobacteria|c__Gammaproteobacteria CW 9.365e-12 4.628

We can also plot the abundance of taxa detected using LEfSe. If you are interested in the taxonomic tree, you can use metacoder package[Foster_Metacoder_2017] to plot the taxonomic tree based on the selected taxa. We do not show the usage here.

The third approach is called rf, which depend on the random forest[11, 12] and the non-parametic test.

trans_env class

　The environmental variables or physicochemical measurements are very useful in analyzing microbial community structure and assembly mechanisms. We first show the RDA analysis using both the dbrda and rda.

Mantel test can be used to check whether there is significant correlations between physicochemical variables and distance metrics.

variable_name cor_method corr_res p_res significance
Temperature pearson 0.452 0.001 ***
Precipitation pearson 0.2791 0.001 ***
TOC pearson 0.13 0.003 **
NH4 pearson -0.05539 0.922
NO3 pearson 0.06758 0.049 *
pH pearson 0.4085 0.001 ***
Conductivity pearson 0.2643 0.001 ***
TN pearson 0.1321 0.002 **

The correlations between physicochemical variables and taxa are important in analyzing or inferring the factors affecting community structure. In this example, we first perform the differential abundance test and random forest analysis to obtain the important genus. Then we use those taxa to perform correlation analysis.

Then, we can plot the correlation results using ggplot2 or pheatmap.

If you are concerned with the relationship between environmental factors and alpha diversity, you can also use this function.

trans_network class

　Network has been a frequently used approach to study the co-occurrence patterns in microbial ecology[13–15]. In this part, we describe all the core contents in the trans_network class. The network construction approaches can be classified into two types: correlation-based and non correlation-based. Several approaches can be used to calculate correlations and significances. So we create a class called trans_corr to calculate correlations. trans_network class inherits the trans_corr class.

We first introduce the correlation-based network. The parameter cal_cor in trans_network controls the correlation calculation method.

Vertex 1766
Edge 17940
Average_degree 20.32
Average_path_length 9.882
Network_diameter 17
Clustering_coefficient 0.6454
Density 0.01151
Heterogeneity 0.9755
Centralization 0.04628
  z module p taxa_roles degree betweenness
OTU_236 0.6822 M1 0 Peripheral nodes 44 59
OTU_32 -0.4248 M1 0 Peripheral nodes 22 117
OTU_2733 -0.9783 M1 0 Peripheral nodes 11 23664
OTU_4050 0.8332 M1 0 Peripheral nodes 47 12753
OTU_52 0.33 M1 0.1352 Peripheral nodes 37 2466
OTU_36 1.034 M1 0 Peripheral nodes 51 3497
OTU_2721 -0.5255 M1 0.4444 Peripheral nodes 20 30
OTU_192 -1.28 M1 0.375 Peripheral nodes 5 0
OTU_94 -1.431 M1 0 Peripheral nodes 2 0
OTU_5862 -1.381 M1 0 Peripheral nodes 3 212

The subset_network() function can be used to take a part of nodes and edges among these nodes from the network. In this function, you should provide the nodes you need using the node parameter.

We also introduce another network construction approach: Probabilistic Graphical Models (PGM), which is implemented in julia package FlashWeave[16]. So if you want to use this method like the following code, you should first install julia language in your computer and the FlashWeave package, and add the julia in the computer path.

trans_nullmodel class

　To make the use of null model convenient, we incorporate several null models and phylogenetic analysis[17]. We first check the phylogenetic signal.

betaNRI(ses.betampd) is used to show the ‘basal’ phylogenetic turnover[18].

If we want to plot the betaNRI, we can use plot_group_distance function in trans_beta class. The results showed that the mean betaNRI of TW is extremely and significantly larger that those in CW and IW, revealing that the basal phylogenetic turnover in TW is high, if no strong dispersal limitation exists among the wetlands of different areas in China.

Sometimes, if you want to perform null model analysis for each group individually with a specific hypothesis, you can calculate the results for each group, respectively. We can find that, when we perform betaNRI for each group respectively, the mean betaNRI between CW and TW are not significantly different, and are both significantly higher than that in IW, revealing that the strength of variable selection in CW and TW may be similar under the condition that each area have a specific species pool.

betaNTI(ses.betamntd) can be used to indicate the phylogenetic terminal turnover.

1 2 3 4 5
0 -5.637 -5.701 -5.758 -5.531
-5.637 0 -6.101 -6.275 -6.013
-5.701 -6.101 0 -6.333 -6.197
-5.758 -6.275 -6.333 0 -6.141
-5.531 -6.013 -6.197 -6.141 0

rcbray can be calculated use function cal_rcbray().

As an example, we also calculate the propotion of the inferred processes on the community assembly.

process percentage
variable selection 4.419
homogeneous selection 48.71
dispersal limitation 0
homogeneous dispersal 8.739
drift 38.13

trans_func class

　Ecological researchers are usually interested in the the funtional profiles of microbial communities. Because functional or metabolic data are more powerful to explain the structure and dynamics of microbial communities and to infer the underlying mechanisms. As metagenomic sequencing is complicated and expensive, using amplicon sequencing data to predict functional profiles is a good choice. Several software are often used for this goal, such as PICRUSt[19], Tax4Fun[20] and FAPROTAX[21, 22]. These tools are great to be used for the prediction of functional profiles based on the prokaryotic communities from sequencing results. Sometimes, it is also important to obtain the functions of each taxa or OTU, not just the whole profile of communities. But it is hard to know which OTU have what functions exactly. FAPROTAX database is a collection of the traits and characteristics of prokaryotes based on the books and literatures. We use it to analyze the biogeochemical roles of prokaryotes in this part.

  methanotrophy acetoclastic_methanogenesis
OTU_4272 0 0
OTU_236 0 0
OTU_399 0 0
OTU_1556 0 0
OTU_32 0 0

The percentages of the OTUs having one trait can reflect the functional redundancy and potential of this function in the community or the specific pattern of the modules in the network.

methanotrophy acetoclastic_methanogenesis
0.39 0.04
0.27 0
0.48 0
0.48 0
0.56 0

We also calculate the functional potential of communities on the biogeochemical cycles by using FAPROTAX software.

  1 2 3
methanotrophy 26 15 16
acetoclastic_methanogenesis 2 0 0
methanogenesis_by_disproportionation_of_methyl_groups 5 0 0
methanogenesis_using_formate 0 0 0
methanogenesis_by_CO2_reduction_with_H2 23 2 5
methanogenesis_by_reduction_of_methyl_compounds_with_H2 0 0 0
hydrogenotrophic_methanogenesis 23 2 5
methanogenesis 25 2 5
methanol_oxidation 12 2 6
methylotrophy 38 17 22

Tax4Fun have a strict input file demand on the taxonomic information. To use Tax4Fun to analyze the trimmed or changed OTU data in R, we provide a link to the Tax4Fun functional prediction.

K00001; alcohol dehydrogenase [EC:1.1.1.1] K00002; alcohol dehydrogenase (NADP+) [EC:1.1.1.2]
0.0004823 5.942e-06
0.0005266 4.017e-06
0.0005054 6.168e-06
0.0005109 5.888e-06
0.0005083 5.547e-06

Now, we use pathway file to analyze the abundance of pathway.

microtable class: sample_table have 90 rows and 4 columns otu_table have 284 rows and 90 columns tax_table have 341 rows and 3 columns

Now, we need to trim data and calculate abundance.

microtable class: sample_table have 90 rows and 4 columns otu_table have 284 rows and 90 columns tax_table have 284 rows and 3 columns Taxa abundance: calculated for level_1,level_2,level_3

Then, we can plot the abundance.

We can also do something else. For example, we can use lefse to test the differences of the abundance and find the important enriched pathways across groups.

Something important

clone

R6 class has a special copy mechanism different from S3 and S4. If you want to copy an object completely, you should use function clone

[1] FALSE

[1] TRUE

change object

All the classes were set public, meaning that you can change, add or remove the objects stored in them as you want.

Contact

If the reader has any question, feel free to join the QQ group: 277434916 for the discussion.

References

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